Stempor, Przemyslaw et al. (2015) International Worm Meeting "SeqPlots - a fast interactive web tool for visualizing next generation sequencing signals along genomic features."

Ahringer, Julie, Stempor, Przemyslaw

[International Worm Meeting, 2015]

Sequencing (NGS) based experiments (e.g. ChIP-seq) generate vast amounts of data, which are driving advances in functional genomics research. To analyse genomic data sets, signal intensities across selected sets of genomic regions are often examined by generating average plots and heatmaps. We found that existing plotting methods were slow or laborious, inhibiting users from producing a large number of plots for data exploration.Here we present SeqPlots, a software that rapidly generates linear profile plots and heatmaps for exploratory data analyses and publications. SeqPlots can plot any set of experimental or in silico produced data (ChIP- and RNA-seq, chromatin accessibility assays, density of sequence motifs, nucleosome occupancy predictions, etc.) over one or multiple sets of genomic features, e.g. genes, promoters, peak calls. After uploading input files users choose any combination of signals and features. SeqPlots precalculates and stores a numeric array of each signal over each feature and presents a clickable array of signal/feature pairs that can be plotted individually or in any combination in a matter of seconds. Average signal plots can be displayed with error estimates and heatmaps can be sorted or clustered using a choice of three clustering algorithms: (1) k-means, (2) hierarchical clustering and (3) self-organizing maps. Clustering is particularly useful for exploratory analyses of profiles and pattern discovery.SeqPlots is designed to be an efficient and user-friendly plotting tool. SeqPlots brings the powerful data analyses and visualization capabilities of the R environment as an easy, intuitive web application, which eliminates the need for a user to have training in programing. Hence, our software significantly shortens the time between retrieving sequencing data and extracting the biological knowledge.SeqPlots is available as a Mac OS X bundle that runs as a desktop application and as an R package from the Bioconductor repository (http://www.bioconductor.org/packages/release/bioc/html/seqplots.html). SeqPlots can also be deployed as a server application for data sharing and remote work. SeqPlots is an active, open development project with issue tracker, wiki pages and OS X app download page hosted by GitHub (https://github.com/przemol/seqplots).

Evans, Kenneth J et al. (2015) International Worm Meeting "Genome organization revealed through chromatin state mapping."

Down, Thomas A, Ahringer, Julie, Stempor, Przemyslaw, Evans, Kenneth J, Chesney, Michael A

[International Worm Meeting, 2015]

Eukaryotic genomes are organized into domains of differential structure and activity. Through chromatin state mapping using 17 chromatin marks in L3 larvae, we identify 20 states that correlate with many biological features as seen previously in other systems. Investigating patterns of states on a larger scale, we observe extended active and inactive domains corresponding with regions of low and high H3K27me3, and we characterize two types of border regions separating domains. Marked borders are associated with higher levels of H3K36me3 and germline active chromatin. These borders show spreading of H3K27me3 into neighboring active regions in animals with reduced H3K36me3 germline marking, suggesting that germline events contribute to domain definition. Regulatory borders are enriched for characteristics of transcriptional regulatory regions. The domain organization shows similarities to topological domains in humans and Drosophila, where insulator proteins such as CTCF show enrichment at boundaries. Using large modENCODE and ENCODE collections of transcription factor binding data, we find that the binding of most transcription factors is enriched at C. elegans borders and Drosophila and human topological boundaries, indicating that the previously observed topological boundary enrichment of insulator proteins is not specific to these factors. We propose that these regulatory regions may help to organize chromatin into domains.

Pokhrel, Bharat et al. (2017) International Worm Meeting "Characterising C. elegans COMPASS complex in gene expression."

Chen, Ron, Appert, Alex, Pokhrel, Bharat, Clifford, Rosamund, Chen, Yannic, Ahringer, Julie, Dong, Yan, Stempor, Przemyslaw

[International Worm Meeting, 2017]

Trimethylation of histone lysine 4 (H3K4me3) is regarded as an active promoter mark across all eukaryotes. H3K4me3 is mainly deposited by an evolutionarily conserved COMPASS complex that contains two key subunits: SET-2, H3K4me3/2 methyltransferase, and CFP-1, a conserved CpG binding protein. Perturbation of this mark has been found to be associated with different diseases, development defects, and incorrect cell fate specification. However, it is unclear whether H3K4me3 play an instructive role in transcription or just a consequence of gene expression. To address this question, we phenotypically characterised and compared cfp-1(tm6369) mutants with a reported loss-of-function set-2(bn129) mutant allele. We observed dramatic loss of H3K4me3, poor fertility, slow growth and ectopic gene expression in both cfp-1 and set-2 mutants. We found that the role of H3K4me3 is to fine tune gene induction, likely by modulating other histone modifications that play an essential role in gene activation. Our results shed light on the role of H3K4me3 in chromatin regulation and function.

Chen, Ron et al. (2015) International Worm Meeting "The roles of CpG density and H3K4me3 at C. elegans promoters."

Chen, Ron, Janes, Jurgen, Dong, Yan, Zeiser, Eva, Gemma, Carolina, Stempor, Przemyslaw, Ahringer, Julie, Down, Thomas, Feuer, Sky

[International Worm Meeting, 2015]

A hallmark of most vertebrate promoters is their association with non-methylated CG-dense sequences (CpG islands), while methylation of the more sparsely distributed CpGs in the remainder of the genome is thought to contribute to transcriptional repression. Non-methylated CG dinucleotides are recognized by the CXXC finger protein 1 (CXXC1/CFP1) which is part of the COMPASS complex. In the complex, the Set1 methyltransferase (SET-2 in C. elegans) generates H3K4me3, a mark of active promoters. Promoter CpGs were thought to be either absent or irrelevant in invertebrates that lack DNA methylation, such as C. elegans. However, a C. elegans CXXC1 ortholog (CFP-1) is present and required for the generation of H3K4me3 (1,2). We found that C. elegans CFP-1 targets promoters with high CpG density and that these promoters are marked by high levels of H3K4me3. Furthermore, as for mammalian promoters, high promoter CpG content in C. elegans is associated with nucleosome depletion irrespective of transcriptional activity. We further show that highly occupied target (HOT) regions identified by the binding of a large number of transcription factors are CpG-rich promoters in C. elegans and human genomes, suggesting that the unusually high factor association at HOT regions may be a consequence of CpG-linked chromatin accessibility. Our results indicate that non-methylated CpG-dense sequence is a conserved genomic signal that promotes an open chromatin state, and is targeted by a CXXC1 ortholog and H3K4me3 modification in both C. elegans and human genomes. To understand the function of CFP-1 and COMPASS/H3K4me3, we are studying a cfp-1 deletion mutant. This mutant has nearly undetectable H3K4me3 and has defects in fertility and the repression of somatic genes in the germline, similar to the phenotype of set-2 mutants (3). Profiling RNA and chromatin in cfp-1 mutants uncovered alterations in histone modifications, chromatin accessibility, and patterns of gene expression. Our results shed light on the mechanism of action and role of CFP-1/H3K4me3 in chromatin regulation and function.References: (1) Simonet et al., 2007, Dev. Biol. 312, 367-83. (2) Li and Kelly, 2011, PLoS Genet. 7, e1001349 (3) Robert et al., 2014, Cell Reports 9, 443-450.

Gaarenstroom, Tessa et al. (2017) International Worm Meeting "Heterochromatin factors collaborate with small RNA pathways to combat repetitive elements and germline genotoxic stress."

Huang, Ni, Wysolmerski, Brian, Ahringer, Julie, McMurchy, Alicia, Appert, Alex, Aussianikava, Darya, Miska, Eric, Sapetschnig, Alexandra, Gaarenstroom, Tessa, Dong, Yan, Stempor, Przemyslaw, Kolasinska-Zwierz, Paulina

[International Worm Meeting, 2017]

Repetitive sequences derived from transposons make up a large fraction of the genome and must be silenced to protect genome integrity. Silencing of these elements is especially important in the germ line, where the piRNA pathway has been shown to be involved. Repeats are often found in heterochromatin, which in C. elegans are regions dispersed over the chromosome arms, however the roles and interactions of heterochromatin proteins are poorly understood. We have shown that a diverse set of heterochromatin factors act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2 and the H2K9me2 histone methyltransferase MET-2/SETDB1 show genome-wide co-binding and enrichment at repetitive elements, and mutants show a derepression of a subset of transposons. Furthermore, heterochromatin mutants are characterized by functionally redundant and temperature sensitive sterility, and display increased germline apoptosis and activation of DNA damage signalling. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting expression of MIRAGE1 DNA transposons or endogenous meiotic double strand breaks. Loss of CEP-1/p53 also ameliorates both their fertility and somatic defects, suggesting that DNA damage signalling contributes to the phenotypes observed. Through genetics and transcriptional profiling, we uncovered complex interactions between heterochromatin factors and the piRNA and nuclear RNAi pathways, including functional redundancy in repetitive element repression between let-418 and nrde-2. This redundancy underlies the importance of safeguarding the genome through multiple means. It is also becoming evident that heterochromatic silencing occurs through different mechanisms at different genomic elements. We are currently dissecting interactions between the various factors, as well as H3K9me2/3, to uncover their mechanisms of action at heterochromatic regions, as well as investigating their roles in development and adult homeostasis.

Gal, C et al. (2015) International Worm Meeting "Regulation of gene expression by the DREAM complex."

Latorre, I, Appert, A, Chesney, M, Ahringer, J, Gal, C

[International Worm Meeting, 2015]

The conserved eight subunit DREAM (DP, Retinoblastoma [Rb]-like, E2F, and MuvB) complex is a regulator of cell cycle and quiescence genes and plays an important role in development and disease. The C. elegans DREAM members are LIN-35/Rb-like, EFL-1/E2F, DPL-1/DP1, LIN-8, LIN-37, LIN-52, LIN-53, and LIN-54. Recent work has demonstrated that C. elegans direct DREAM targets are de-repressed in the absence of the Rb-like protein LIN-35 and that high gene body levels of the histone variant H2A.Z are linked with target repression1. However, the mechanism that generates gene body H2A.Z enrichment, and the relationship between DREAM and H2A.Z are not currently understood. Furthermore, the functions and regulatory interactions of individual complex members are not clear. For example, LIN-35 has been demonstrated to be a transcriptional repressor by a number of groups, whilst the DP and E2F proteins have previously been reported to act as activators of transcription2. In addition, mutants of individual DREAM complex members do not all have the same phenotype, indicating different functions. Therefore, we are investigating the contribution of DPL-1 (DP) and EFL-1 (E2F) to DREAM target gene repression. We are also carrying out a GFP reporter screen to identify additional components required for DREAM-mediated repression, to elucidate the mechanisms behind DREAM mediated developmental control.1. Latorre, I., Chesney, M.A., Garrigues, J.M., Stempor, P., Appert, A., Francesconi, M., Strome, S., and Ahringer, J. (2015) 'The DREAM complex promotes gene body H2A.Z for target repression', Genes Dev., 29, 495-5002. Chi, W & Reinke, V. (2006) 'Promotion of oogenesis and embryogenesis in the C. elegans gonad by EFL-1/DPL-1 (E2F) does not require LIN-35 (pRB)', Development, 133, 3147-57.